What every physician needs to know:

The disease originates from a multipotent hematopoietic stem cell that acquires a mutation of the PIG-Agene. Expansion and differentiation of the PIG-A mutant stem cell leads to clinical manifestations of the disease. The PIG-Agene product is required for the biosynthesis of glycophosphatidylinositol (GPI) anchors, a glycolipid moiety that attaches dozens of proteins to the plasma membrane of cells. Consequently, the PNH stem cell and all of its progeny have a reduction or absence of GPI anchored proteins. Two of these proteins, CD55 and CD59, are complement regulatory proteins. CD55 inhibits C3 convertases and CD59 blocks formation of the membrane attack complex (MAC) by inhibiting incorporation of C9 into the MAC.

The loss of complement regulatory proteins renders PNH erythrocytes susceptible to both intravascular and extravascular hemolysis, but it is the intravascular hemolysis that contributes to much of the morbidity and mortality from the disease. Intravascular hemolysis releases free hemoglobin into the plasma. Free plasma hemoglobin scavenges nitric oxide, and depletion of nitric oxide at the tissue level contributes to numerous PNH manifestations, including esophageal spasm, male erectile dysfunction, renal insufficiency, and thrombosis.

The natural history of PNH is highly variable, ranging from indolent to life-threatening. Eculizumab is a monoclonal antibody that blocks terminal complement activation and is effective in treating some forms of PNH.

Are you sure your patient has paroxysmal nocturnal hemoglobinuria? What should you expect to find?

Patients with PNH have signs and symptoms of intravascular hemolysis. These patients tend to have a normocellular to hypercellular bone marrow with erythroid hyperplasia, an elevated reticulocyte count, a large population of PNH cells (usually greater than 60% PNH granulocytes), and an LDH (lactate dehydrogenase) that is two to ten times the upper limit of normal. Hemoglobinuria, smooth muscle dystonias (for example, esophageal spasm and erectile dysfunction), severe fatigue, dyspnea, and thrombosis are common in patients with PNH. Patients with these signs and symptoms are often referred to as having “classical” PNH.

An expanded PNH clone is also found in up to 70% of patients with acquired aplastic anemia (AA), demonstrating a pathophysiological link between these disorders. In contrast to patients with classical PNH, these patients typically have a lower percentage of PNH cells and are often referred to as having AA/PNH. Typically, the number of glycosylphosphatidyl-inositol anchored protein (GPI-AP) deficient granulocytes detected in AA patients is fewer than 10% at diagnosis, but occasional patients may have larger clones. Although most AA patients exhibit no signs or symptoms of PNH early in the natural history of their disease when the PNH clone size is small, many, but not all, will progress to classical PNH.

- PNH patients more commonly experience venous thrombosis, but arterial thrombosis may occur. Thrombosis may occur in large and small vessels. Common sites of thrombosis include abdominal veins (hepatic, portal, splenic, mesenteric), cerebral veins, and dermal veins. Thrombosis is more common in patients with more than 50% PNH granulocytes.

Abdominal pain

- May result from thrombosis or spasm.

Esophageal spasm

- Patients with large PNH clones may complain of dysphagia or odynophagia; this is due to scavenging of nitric oxide by the release of free hemoglobin.

Erectile dysfunction

- Also a direct consequence of nitric oxide scavenging. Also more common in patients with large PNH clones.

Fatigue

- May be out of proportion with the degree of anemia.

Beware of other conditions that can mimic paroxysmal nocturnal hemoglobinuria:

Other conditions that can mimic PNH are:

Acquired aplastic anemia

Myelodysplastic syndromes

Other hemolytic anemias

Which individuals are most at risk for developing paroxysmal nocturnal hemoglobinuria:

PNH is an extremely rare condition. The precise incidence is unknown, but it is estimated to be roughly 1 to 5 per million people. PNH and acquired aplastic anemia, are closely related diseases. The risk for developing PNH in patients with acquired aplastic anemia is 20-30%. Moreover, 40-70% of patients with acquired aplastic anemia harbor a small to moderate PNH population at diagnosis.

What laboratory studies should you order to help make the diagnosis and how should you interpret the results?

Peripheral blood flow cytometry demonstrating an absence of GPI anchored proteins on two or more hematopoietic lineages is required to establish a diagnosis of PNH.

PNH flow cytometry should be performed on peripheral blood and not bone marrow. Guidelines for the diagnosis and monitoring of PNH have recently been published. Ideally, at least two different monoclonal antibodies, directed against two different GPI anchored proteins, on at least two different cell lineages should be used to diagnose a patient with PNH.

Monoclonal antibodies directed against the GPI anchored proteins CD59, CD55, CD14, CD24 and others are commonly used. A fluorescein-labeled proaerolysin variant (FLAER), is increasingly being used as a flow cytometric assay to diagnose PNH. FLAER binds selectively and with high affinity to the GPI anchor. Since the GPI anchor is the major determinant for binding FLAER, it allows for the direct assessment of GPI anchor expression on most cell lineages. Red cells and platelets are notable exceptions.

How to interpret PNH flow cytometry

Assessment of PNH populations in leukocytes (usually granulocytes or monocytes) is the optimal method for assessing the size of the PNH clone. Assessment of red cells should also be performed, but testing of red cells alone is not adequate for evaluation of PNH patients, because the short life span of PNH erythrocytes and blood transfusions from healthy donors lead to an underestimation of the PNH clone.

Patients with PNH typically have a large percentage (40 to 99%) of PNH granulocytes. Patients with AA usually have a relatively small percentage (0.1 to 10%) of PNH granulocytes. However, patients with aplastic anemia may experience clonal expansion of the PNH cells leading to an overlap condition referred to as AA/PNH.

GPI-AP deficient cells (usually less than 1% of granulocytes) have also been reported in patients with myelodysplastic syndrome (MDS). PIGAmutant blood cells are readily detected in the blood and bone marrow of healthy control subjects at a frequency of roughly 1 in 50,000 (0.002%). Meticulous molecular and statistical analysis reveals that unlike PIG-A mutations in PNH, most if not all of these mutations appear to arise from colony forming cells and not multipotent hematopoietic stem cells. PNH clones of less than 0.01% should be viewed with caution, given this background rate of GPI-AP deficient cells, in the blood of healthy subjects.

Tests required

Complete blood count with differential

- Virtually all patients will have depression of at least one hematopoietic lineage (anemia, thrombocytopenia, or neutropenia).

Reticulocyte count

- Usually elevated in patients with classical PNH, but often low in patients with AA/PNH.

LDH

- Often markedly elevated (five to ten times the upper level of normal) in patients with classical PNH. May be normal or only mildly elevated in patients with a granulocyte clone size of less than 30%.

D-dimer

- Elevated in patients with ongoing thrombus formation. Elevated levels should prompt further clinical evaluation for thrombosis if clinically indicated and consideration for anticoagulation.

Bone marrow aspirate, biopsy, iron stain, and cytogenetics

- Patients with classical PNH usually have a normocellular or hypercellular bone marrow with erythroid hyperplasia. Stainable iron is often absent. Erythroid dysplasia due to the robust red cell turnover is not uncommon in PNH. A small percentage of PNH patients will transform to MDS or acute myeloid leukemia; thus, karyotypic abnormalities are found in a small percentage of patients.

- Indirect bilirubin and elevated SGOT are common in patients with intravascular hemolysis. Elevation of the direct bilirubin (especially in conjunction with ascites) should lead to evaluation of hepatic and portal veins with ultrasound, computed tomography (CT) scan or magnetic resonance imaging (MRI).

Renal function (blood urea nitrogen [BUN] and creatinine)

- Usually normal, but acute and chronic renal failure may occur in patients with large amounts of intravascular hemolysis.

Serum iron/total iron binding capacity/serum ferritin

- May be useful to help assess iron stores which may be reduced, due to intravascular hemolysis.

What imaging studies (if any) will be helpful in making or excluding the diagnosis of paroxysmal nocturnal hemoglobinuria?

Imaging studies are not routinely performed. However, in patients describing intractable headache or severe abdominal pain, with or without increasing abdominal girth, imaging studies to help rule out thrombosis may be indicated. Abdominal ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) (listed least to most sensitive), can be used to detect abdominal vein thrombosis. CT or MRI may be used to detect intracranial thrombosis.

If you decide the patient has paroxysmal nocturnal hemoglobinuria, what therapies should you initiate immediately?

For patients with classical PNH, allogeneic bone marrow transplantation (BMT) and complement inhibition with eculizumab are the only established therapies. Corticosteroids may improve hemoglobin levels and reduce hemolysis is some PNH patients, but the long term toxicity and limited efficacy makes these agents unappealing.

Asymptomatic patients and patients with mild symptoms do not require immediate treatment. In classical PNH, eculizumab therapy should be considered for patients with disabling fatigue, thromboses, transfusion dependence due to primarily to hemolysis, frequent pain paroxysms, worsening renal insufficiency, or other end organ complications from disease. Watchful waiting is appropriate for asymptomatic patients or those with mild symptoms.

Patients who meet criteria for severe aplastic anemia with a PNH clone (AA/PNH) should be managed with either allogeneic BMT or immunosuppressive therapy, depending upon the age of the patient and the availability of a suitable stem cell donor.

Supportive care: Loss of iron through the urine may be significant in PNH, thus supplement iron should be administered to patients who are iron deficient. Supplemental folic acid (1 to 2mg daily) is also recommended, as with other chronic hemolytic anemias. Red cell and platelet transfusions should be administered if clinically indicated. Erythropoietin is not effective in improving the anemia of PNH in most cases.

More definitive therapies?

Complement inhibition

Eculizumab is a humanized monoclonal antibody against complement component 5 (C5) that inhibits terminal complement activation. Since C5 is common to all pathways of complement activation, blockade at this point aborts progression of the cascade regardless of the stimuli. Moreover, prevention of C5 cleavage blocks the generation of the potent proinflammatory and cell lytic molecules C5a and C5b-9, respectively. Importantly, C5 blockade preserves the critical immunoprotective and immunoregulatory functions of upstream components that culminate in C3b-mediated opsonization and immune complex clearance. In 2007, the United States Food and Drug Administration approved eculizumab for use in PNH; this was based upon its efficacy in two phase III clinical trials.

Eculizumab is highly effective in reducing intravascular hemolysis in PNH; it does not stop extravascular hemolysis and it does not treat bone marrow failure. Thus, eculizumab is most effective in patients with classical PNH. Treatment with eculizumab decreases or eliminates the need for blood transfusions, improves quality of life, and reduces the risk of thrombosis. Before starting therapy, all patients should be vaccinated against Neisseria meningitides because inhibition of complement at C5 increases the risk for developing infections with encapsulated organisms, particularly N. meningitides and Neisseria gonorrhoeae.

Eculizumab is administered intravenously at a dose of 600mg weekly for the first 4 weeks. On week 5, the dose is increased to 900mg intravenously, and thereafter the drug is dosed at 900mg intravenously every 14 + 2 days. Eculizumab is safe and well-tolerated, but must be continued indefinitely, since it does not treat the underlying cause of the disease. The most common side-effect, headache, occurs in roughly 50% of patients, after the first dose or two, but rarely occurs thereafter.

Neisserialsepsis is the most serious complication of eculizumab therapy; thus, it is imperative to remind patients that they have a 0.5% yearly risk of acquiring Neisserial sepsis, even after.

vaccination. Moreover, patients should be revaccinated against Neisseriameningitidis every 3 to 5 years after starting eculizumab. Eculizumab should be used judiciously, due to its high cost.

Monitoring patients on eculizumab

Most patients notice symptomatic improvement within hours to days after the first dose of eculizumab. Patients should be monitored with a complete blood count, reticulocyte count, LDH, and biochemical profile weekly for the first 4 weeks and then at least monthly thereafter. The LDH usually returns to normal or near normal within days to weeks after starting eculizumab; however, the reticulocyte count usually remains elevated and the hemoglobin response is highly variable. The reticulocyte count often remains elevated because most PNH patients on eculizumab continue to have some extravascular hemolysis.

PNH erythrocytes frequently have increased deposition of C3 fragments, due to CD55 deficiency and these cells are prematurely removed by the spleen. The hemoglobin response is largely dependent upon the degree of extravascular hemolysis and the amount of underlying bone marrow failure.

In classical PNH patients who are transfusion dependent, a marked decrease in red cell transfusions is observed in most patients, with over 70% achieving transfusion independence.

Breakthrough intravascular hemolysis and a return of PNH symptoms occurs in less than 5% of PNH patients treated with eculizumab. This typically occurs 1 or 2 days before the next scheduled dose, and is accompanied a spike in the LDH level. If this occurs on a regular basis, the interval between dosing can be shortened to 12 or 13 days, or the dose of eculizumab can be increased to 1200mg every 14 days.

It is also important to recognize that increased complement activation that accompanies infections (for example, influenza or viral gastroenteritis) or trauma can also result in transient breakthrough hemolysis. These single episodes of breakthrough hemolysis do not require a change in dosing.

Bone marrow transplantation

BMT is the only curative therapy for PNH and has been shown to eradicate the PNH clone in patients with classical PNH and AA/PNH; however, it is associated with significant morbidity and mortality. The use of allogeneic BMT to treat PNH has decreased since the introduction of effective drug therapy (eculizumab), but indications for BMT still exist.

Most investigators agree that BMT should not be offered as initial therapy for most patients with classical PNH, given the transplant related morbidity/mortality, especially when using unrelated or mismatched donors. Exceptions are PNH patients in countries where eculizumab is not available.

BMT is also a reasonable option for patients who do not have a good response to eculizumab therapy. AA/PNH patients continue to be reasonable candidates for BMT if they have life threatening cytopenias. Similar to other BMT patients, younger age and the availability of a fully matched sibling donor are favorable prognostic factors.

It is now clear that a myeloablative conditioning regimen is not required to eradicate the PNH clone. Allogeneic BMT, following non-myeloablative conditioning regimen can cure PNH. Whether or not there is an advantage to non-myeloablative transplants in PNH will require further study; however, nonmyeloablative regimens may be preferable in young patients seeking to maintain fertility, or patients with moderate organ dysfunction who may not tolerate a myeloablative regimen.

What other therapies are helpful for reducing complications?

Thrombosis is the leading cause of death from PNH and should be treated promptly with anticoagulation and sometimes thrombolytic therapy, depending on the location of the thrombus. Anticoagulation is only partially effective in preventing thrombosis in PNH, thus thrombosis in PNH is a strong indication for initiating treatment with eculizumab.

A more controversial issue is whether PNH patients not taking eculizumab should receive prophylactic anticoagulation, and whether patients on eculizumab therapy who have had a prior thrombus, need to remain on anticoagulation. Prophylactic anticoagulation has never been proven to prevent thrombosis in PNH patients and is often dangerous given the low platelet counts that are observed in many PNH patients.

Discontinuing anticoagulation in patients on eculizumab with a previous thrombosis is even more controversial and there are insufficient data to make strong recommendations.

What should you tell the patient and the family about prognosis?

The natural history of PNH ranges from indolent to severely debilitating, and life-threatening. Females and males are equally affected with the median age of diagnosis being 40 years old. The median survival from time of diagnosis is 15 to 20 years. Thrombosis, severe pancytopenia, evolution to MDS or leukemia, older age, and thrombocytopenia at diagnosis portend a poor prognosis.

Older literature, where patients were diagnosed with PNH based on the Ham test or sucrose hemolysis test, reported on the occurrence of spontaneous long-term remissions in up to 10% of PNH cases; however, in patients diagnosed with PNH based on flow cytomeric assays, spontaneous remissions are rare.

These natural history studies were all performed before the introduction of eculizumab. While eculizumab has not yet been demonstrated to change the natural history of PNH, the drug appears to markedly decrease the risk for thrombosis, the leading cause of death from PNH.

“What if” scenarios.

What if my PNH patient becomes pregnant?

PNH patients who become pregnant have an increased risk of complications in both the mother and the fetus. Besides thrombotic events, complications may include infections, bleeding, anemia, and an increased risk of miscarriages, fetal death, and prematurity. Transfusion requirements are likely to increase. Thus, all pregnant PNH patients should be closely monitored during pregnancy by both hematology and obstetric specialists.

In view of the high risk for thrombosis, the use of low molecular weight heparin should be considered as soon as pregnancy is confirmed. Anticoagulation should be continued until at least 3 months after delivery. There is limited experience with using eculizumab in pregnancy; however, eculizumab does not appear to cross the placenta efficiently or be excreted in breast milk. It is important to recognize that the dose of eculizumab may need to be increased during the third trimester if patients begin to experience hemoglobinuria and a rising LDH in between doses.

What if my PNH patient on eculizumab is still requiring blood transfusions?

Most patients managed with complement blockade will have significant medical improvement, but up to 30% will still need occasional blood transfusions. Even patients who become transfusion independent will often continue to have a mild to moderate anemia and an elevated reticulocyte count. This is due to C3 fragment deposition on the PNH erythrocytes and subsequent extravascular hemolysis in the spleen. While eculizumab is quite effective at mitigating the CD59 deficit that leads to intravascular hemolysis, it does not reduce C3 fragment deposition that is a direct consequence of the CD55 deficiency (see mechanism of hemolysis below).

Other mechanisms of anemia to be considered in PNH patients on eculizumab are bone marrow failure due to concomitant aplastic anemia and insufficient complement blockade from the eculizumab. Most of these etiologies can be distinguished by ordering the following laboratory tests: complete blood count, reticulocyte count, and LDH 2 to 7 days after the last dose of eculizumab and immediately before the next scheduled dose.

What if my patient develops liver disease?

Hepatic vein thrombosis (Budd-Chiari syndrome) is a common site of thrombosis in PNH and is frequently a fatal complication. Moreover, PNH is probably the condition that confers the highest risk for developing hepatic vein thrombosis. The clinical manifestations of hepatic vein thrombosis include abdominal pain, hepatomegaly, jaundice, ascites, and weight gain. The onset of symptoms may be abrupt or insidious.

The best non-invasive tests to confirm the diagnosis include CT scanning, MRI, and ultrasonography. Thrombosis may involve the small hepatic veins, large sized hepatic veins, or both. Thrombolytic therapy has been used successfully to restore venous patency and reverse the hepatic congestion; however, due to the potential danger of this approach, it should be used judiciously. Patients with acute onset disease, preserved platelet counts (greater than 50,000/mm3), and large vessel involvement are the best candidates for thrombolysis.

For patients with massive ascites who are not suitable candidates for thrombolytic therapy, transjugular intrahepatic portal-systemic shunting, or surgical shunting can successfully palliate some patients. Orthotopic liver transplantation has not been recommended, due to the high risk of recurrent hepatic thrombosis following surgery; however, with complement inhibition, it may now be safe to perform liver transplantation in patients with liver failure due to PNH.

Portal vein thrombosis

Portal vein thrombosis is also common in PNH and may occur with or without hepatic vein thrombosis. Patients frequently present with nausea, vomiting, abdominal pain, and liver dysfunction. Management is similar to that of hepatic vein thrombosis.

Patients with bone marrow failure will have a persistently low reticulocyte count, a normal or mildly elevated LDH, and concomitant thrombocytopenia and neutropenia

Patients with significant extravascular hemolysis will have an elevated reticulocyte count and a normal or mildly elevated LDH. Some will develop a positive direct antiglobulin (Coombs) test

- Recommendations: No definitive therapy.

Patients with insufficient complement blockade will have worsening anemia associated with an elevated reticulocyte count and a markedly elevated LDH on the blood draw immediately before their next scheduled dose

- Recommendations: Consider increasing the dose of eculizumab. Some patients will need to be reloaded with the drug.

Pathophysiology

Genetics of PNH

PNH is an acquired clonal hematopoietic stem cell disorder. The human PIG-Agene contains six exons, five introns and extends over 17kb; it codes for a protein that contains 484 amino acids (60kDa). In humans, there is a single copy of the gene located on the short arm of the X chromosome (Xp22.1). Most PIG-A mutations are small insertions or deletions, usually one or two base pairs, that result in a frameshift in the coding region and consequently a shortened, non-functional product.

How do PIG-A mutations cause PNH?

GPI anchor biosynthesis is a post translational event that occurs in the endoplasmic reticulum. More than 20 different genes, and at least 10 steps are involved in making a mature GPI anchor; thus, it was initially surprising to discover that PNH patients virtually always harbor defects in just one of these genes, PIG-A. However, the localization of the PIG-A gene to the X-chromosome and the localization of all other genes involved in GPI anchor biosynthesis to autosomes readily explains this finding. Since males have only one X chromosome and females, through lyonization, have one inactivated X chromosome per cell, a single PIG-Amutation can result in dysfunctional GPI anchored protein synthesis.

Mechanism of hemolysis in PNH?

There are dozens of GPI anchored proteins, and their functions are highly varied. CD59 (membrane inhibitor of reactive lysis) and CD55 (decay accelerating factor) are the most widely expressed GPI anchored proteins, and can be found on all hematopoietic lineages. Their absence on the surface of red cells leads to the intravascular and extravascular hemolysis in PNH. CD59 directly interacts with the membrane attack complex (MAC) to prevent lytic pore formation, by blocking the aggregation of C9. CD55 functions to accelerate the rate of destruction of membrane bound C3 convertase. Hence, CD55 reduces the amount of C3 that is cleaved and CD59 reduces the number of MAC that is formed.

Nitric oxide and PNH?

Nitric oxide (NO) is a major regulator of vascular physiology, and many clinical manifestations of PNH are explained by hemoglobin mediated nitric oxide scavenging. Normally, oxygen and arginine are reacted with nitric oxide synthase (NOS) in the endothelium to produce NO and citrulline. NO then acts on the vascular wall to maintain normal tone and limit platelet activation. Free oxyhemoglobin in the plasma has a very high affinity for NO, and normally the erythrocyte membrane minimizes NO scavenging by oxyhemoglobin.

In PNH, failure of complement regulation on the PNH erythrocyte membrane leads to intravascular hemolysis and the release of large amounts of free hemoglobin into the plasma. Furthermore, intracellular arginase is released. Thus, hemolysis leads to the release of cell-free hemoglobin, which acts as an NO sump, as well as the release of arginase, which decreases the amount of substrate available for NO synthesis. Haptoglobin is one compensatory mechanism for free hemoglobin removal, but the concentration of plasma hemoglobin in PNH is much greater than the capacity of haptoglobin to remove the hemoglobin from plasma.

The depletion of NO contributes to the symptoms of fatigue, abdominal pain, esophageal spasm, erectile dysfunction, and thrombosis that are characteristic of PNH. Indeed, the size of the PNH clone as measured by flow cytometry correlates closely with the risk for hemoglobinuria, thrombosis, erectile dysfunction, abdominal pain, and esophageal spasm; patients with a highly expanded PNH clone (greater than 60% PNH granulocytes) are much more likely to suffer from these complications than patients with a relatively small PNH clone. Further evidence linking free hemoglobin to these complications comes from clinical trials of free hemoglobin blood substitutes; common adverse effects in these studies include pulmonary hypertension, esophageal spasm, and abdominal pain.

What other clinical manifestations may help me to diagnose paroxysmal nocturnal hemoglobinuria?

History

The classic manifestation from which PNH derives its name – paroxysmal bouts of reddish, brownish, or “cola-colored” urine that strikes predominantly overnight, is described by a minority of PNH patients. Most PNH patients have no noticeable hemoglobinuria or have intermittent episodes of hemoglobinuria with no relation to the time of day. Early speculation that the nocturnal hemoglobinuria was a function of a mild drop in pH that occurs with sleep has not been validated.

Physical examination

Patients with PNH often exhibit common findings of hemolytic anemia including pallor and jaundice. Patients with abdominal vein thrombosis may present with abdominal pain, increased abdominal girth, and ascites. Skin manifestations of PNH are rare, but a painful erythematous skin lesion that may develop a black eschar could represent dermal thrombosis. Splenomegaly is rare, but is usually due to splenic vein thrombosis.

What other additional laboratory studies may be ordered?

The original assays to diagnose PNH included the Ham test and the sucrose hemolysis test. These erythrocyte based assays do not reliably quantitate the percentage of PNH cells, and can be falsely negative in patients who have received red cell transfusions.

(First paper from the International PNH Registry. This is a worldwide, observational, non-interventional study collecting safety, effectiveness, and quality-of-life data from patients with a PNH clone.)

(Multinational double-blinded, randomized trial of eculizumab, versus placebo. The paper demonstrates that eculizumab decreases intravascular hemolysis, decreases the need for transfusions and improves quality of life in patients with classical PNH.)